Mario C. De Tullio

Ascorbic acid: much more than just an antioxidant

Vitamin C (ascorbic acid (AA)) is very popular for its antioxidant properties. Consequently, many other important aspects of this multifaceted molecule are often underestimated or even ignored. In the present paper, we have tried to bring to the foreground some of these aspects, including the peculiarities of the AA biosynthetic pathway in different organisms, the remarkable function of AA as a co-substrate of many important dioxygenases, the role of AA-regenerating enzymes and the known pathways of AA catabolism, as well as the intriguing function of AA in gene expression.

The role of ascorbic acid in cell metabolism : between gene-directed functions and unpredictable chemical reactions

Summary Ascorbic acid (AA) acts in the cell as an electron donor in a number of catalysed and non-catalysed reactions. This accounts for its involvement in many different physiological phenomena. Much attention has been directed to the study of non-catalysed ≪unpredictable≫ chemical reactions, in which AA has the general role of ≪antioxidant molecule≫, but this is not sufficient to explain the apparent ≪pleiotropic action≫ of AA. An increasing amount of data supports the view that AA is specifically required for the activity of 2-oxoacid-dependent dioxygenases (2-0DDs), a class of enzymes including those regulating the synthesis of hydroxyproline-containing proteins and hormones in both plants and animals. Understanding that the role of AA in cell metabolism is much wider than generally assumed, will allow researchers to focus on its ≪gene-directed functions≫, leading to actual advances in many fields of biological research.

Changes in onion root development induced by the inhibition of peptidyl-prolyl hydroxylase and influence of the ascorbate system on cell division and elongation

Abstract. Post-translational hydroxylation of peptide-bound proline residues, catalyzed by peptidyl-prolyl-4 hydroxylase (EC 1.14.11.2) using ascorbate as co-substrate, is a key event in the maturation of a number of cell wall-associated hydroxyproline-rich glycoproteins (HRGPs), including extensins and arabinogalactan-proteins, which are involved in the processes of wall stiffening, signalling and cell proliferation. Allium cepa L. roots treated with 3,4-DL-dehydroproline (DP), a specific inhibitor of peptidyl-prolyl hydroxylase, showed a 56% decrease in the hydroxyproline content of HRGP. Administration of DP strongly affected the organization of specialized zones of root development, with a marked reduction of the post-mitotic isodiametric growth zone, early extension of cells leaving the meristematic zone and a huge increase in cell size. Electron-microscopy analysis showed dramatic alterations both to the organization of newly formed cell walls and to the adhesion of the plasma membranes to the cell walls. Moreover, DP administration inhibited cell cycle progression. Root tips grown in the presence of DP also showed an increase both in ascorbate content (+53%) and ascorbate-specific peroxidase activity in the cytosol (+72%), and a decrease in extracellular “secretory” peroxidase activity (−73%). The possible interaction between HRGPs and the ascorbate system in the regulation of both cell division and extension is discussed.

Redox regulation of root apical meristem organization: Connecting root development to its environment

Post-embryonic root growth relies on the proliferative activity of the root apical meristem (RAM), consisting, in part, of cells with juvenile characteristics (stem cells). It is generally, but erroneously held that the RAM indefinitely produces new cells throughout the lifespan of a plant, resulting in indeterminate root growth. On the contrary, convincing data, mainly from the lab of Thomas L. Rost, show in all species analyzed so far, including Arabidopsis, that RAM organization changes over time in parallel with both a cessation of the production of new cells, and a consequent reduction in root growth, even under optimal conditions. In addition, RAM organization evolved to become highly plastic and dynamic in response to environmental triggers (e.g. water and nutrient availability, pollutants). Under unfavourable conditions, the RAM is rapidly reorganized, and, as a result of the cessation of new cell production at the root tip, root growth is altered, and lateral root production is enhanced, thus providing the plant additional strategies to overcome the stress. It is now becoming increasingly clear that this environment-responsive developmental plasticity is linked to reactive oxygen/nitrogen species, antioxidants, and related enzymes, which form part of a complex signalling module specifically operating in the regulation of RAM functioning, in strict relationship with hormonal control of root development exerted by auxin, gibberellins and cytokinins. In turn, such redox/hormone crosstalk regulates gene expression.

Altered stomatal dynamics in ascorbate oxidase over-expressing tobacco plants suggest a role for dehydroascorbate signalling

Control of stomatal aperture is of paramount importance for plant adaptation to the surrounding environment. Here, we report on several parameters related to stomatal dynamics and performance in transgenic tobacco plants (Nicotiana tabacum L., cv. Xanthi) over-expressing cucumber ascorbate oxidase (AO), a cell wall-localized enzyme of uncertain biological function that oxidizes ascorbic acid (AA) to monodehydroascorbic acid which dismutates yielding AA and dehydroascorbic acid (DHA). In comparison to WT plants, leaves of AO over-expressing plants exhibited reduced stomatal conductance (due to partial stomatal closure), higher water content, and reduced rates of water loss on detachment. Transgenic plants also exhibited elevated levels of hydrogen peroxide and a decline in hydrogen peroxide-scavenging enzyme activity. Leaf ABA content was also higher in AO over-expressing plants. Treatment of epidermal strips with either 1 mM DHA or 100 microM hydrogen peroxide resulted in rapid stomatal closure in WT plants, but not in AO-over-expressing plants. This suggests that signal perception and/or transduction associated with stomatal closure is altered by AO over-expression. These data support a specific role for cell wall-localized AA in the perception of environmental cues, and suggest that DHA acts as a regulator of stomatal dynamics.

Dehydroascorbate and dehydroascorbate reductase are phantom indicators of oxidative stress in plants

In many physiological studies dehydroascorbate (DHA) reductase is regarded as one of the chloroplast enzymes involved in the protection against oxidative stress. Here, evidence is presented that plant cells do not possess a specific DHA reductase. The DHA reductase activities measured in plant extracts are due to side reactions of proteins containing redox‐active dicysteine sites. Native gel electrophoresis combined with specific activity staining revealed three different proteins with DHA reductase activity in leaf and chloroplast extracts. These proteins have been identified as thioredoxins and trypsin inhibitors (Kunitz type) by Western blot analysis. The essential regulatory functions of thioredoxins in chloroplast metabolism are strongly inhibited in the presence of as little as 50 μM DHA. Thus, the intracellular DHA concentration should be kept below 50 μM but not all proteins with DHA reductase activity are effective enough for this purpose. A specific DHA reductase is frequently demanded as part of the enzymatic equipment to avoid oxidative stress. We argue that this is not necessary because in chloroplasts DHA does not accumulate to any significant extent due to the high activities of monodehydroascorbate reductase and of reduced ferredoxin.

The ascorbic acid system in seeds: to protect and to serve

The ascorbic acid (ASC) system functions dynamically in seeds, although the strategies for ASC production and utilization may vary according to seed developmental and functional stages. In orthodox seeds, ASC content and ASC peroxidase activity increase during the early stages of development, then decrease during the desiccation stage, so that, at quiescence, seeds have neither ASC nor ASC peroxidase, but retain a small amount of dehydroascorbic acid (DHA) and significant activities of ASC recycling enzymes. ASC and ASC peroxidase activity re-start after a few hours from the onset of imbibition. In contrast, the ASC system is little affected during germination of recalcitrant seeds. Although the presence of the ASC system in seeds has often been considered only within the framework of seed antioxidant defences, ASC function in seeds is also likely to be related to its action as a specific co-substrate required for the activity of dioxygenases (e.g. 1-aminocyclopropane carboxylate oxidase, gibberellic acid hydroxylases and 9-cis-epoxycarotenoid dioxygenases) involved in the synthesis of ethylene, gibberellins and abscisic acid, respectively. The possible role of ASC in coordinating the activities of these key enzymes is discussed.

Dehydroascorbate-reducing proteins in maize are induced by the ascorbate biosynthesis inhibitor lycorine

Abstract Dehydroascorbate (DHA) reductase (glutathaone: dehydroascorbate oxidoreductase, EC 1.8.5.1) has been generally considered a specific enzyme of the ascorbate-glutathione cycle. However, at least four distinct proteins can catalyze in vitro both glutathione-dependent DHA reduction and other reactions mainly related to thiol-disulphide exchange. These data have raised questions both on the existence of specific DHA reductase and the actual physiological role of DHA-reducing proteins (DRP). We have observed characteristic electrophoretic patterns of DRP in dark-germinating embryos of different plant species. Marked differences were observed not only in the number, but also in the migration rate of DRP under non-denaturing conditions. In order to evaluate the actual contribution of DRP activity to ascorbate (ASC) regeneration under conditions limiting ASC biosynthesis, Z. mays germinating embryos excised from endosperm were either incubated in distilled water or treated with the alkaloid lycorine, an inhibitor of ASC biosynthesis. In parallel with the decrease in ASC content, a strong enhancement in DRP activity occurred. The increase in DRP activity was prevented by cycloheximide, and thus seems to be due to de novo protein synthesis. The possible involvement of DRP in avoiding DHA accumulation under adverse environmental conditions is discussed.

Ascorbate oxidase is the potential conductor of a symphony of signaling pathways.

The functional role of ascorbate oxidase (AO; EC 1.10.3.3) has never been fully explained so far, due to the difficulties in understanding the presence of an enzyme specifically oxidizing ascorbate with no obvious advantage, and the apparent disadvantage of lowering plant stress resistance as a consequence of ascorbate consumption. Here we suggest a complete change of perspective, by proposing an essential role of AO as a modulator of both ascorbate and oxygen content, with relevant implications related to signaling. By affecting the overall redox state, AO is actually involved in redox regulation in the extracellular matrix. In addition, AO can contribute to creating a hypoxic microenvironment, especially relevant in the maintenance of meristem identity and the establishment of mutualistic plant-microbe interactions. We also hypothesize the possible involvement of AO in the activation of a signaling cascade analogous to the mechanism of prolyl hydroxylases/Hypoxia Inducible Factors in animals.

Beyond the Antioxidant: The Double Life of Vitamin C

When considering the history of vitamin C, and the names given to this molecule in early days, the Latin proverb nomen est omen suddenly comes to mind. Around 1920, when Casimir Funk introduced the term Vitamin C to indicate the nutritional factor necessary to prevent the pathological state known as scurvy, the nature of the active molecule was still unknown (Davies MB, Austin J, Partridge DA (1991) Vitamin C: Its chemistry and biochemistry. The Royal Society of Chemistry, Cambridge UK). Almost in the same years, Albert Szent-Giorgyi was striving to identify a new 6-carbon sugar he had obtained in crystal form from oranges, lemons, cabbage and adrenal glands. As humorously described by Szent-Giorgyi himself (Szent-Giorgyi A (1963) Lost in the twentieth century. Annu Rev Biochem 36:1–15), he intended to name this yet unknown carbohydrate “ignose”. When this name was rejected by Sir Arthur Harden, editor of the Biochemical Journal, he suggested to name it “godnose”, meaning that only God could know the real identity of the molecule. Obviously, also this choice was considered inappropriate by Harden, who suggested the plain name “hexuronic acid”. Only later, when the structure of “hexuronic acid” had been completely elucidated, and biological tests performed by Swirbely identified this molecule as the anti-scurvy factor vitamin C, Szent-Giorgyi and Walter Norman Haworth decided to eventually name it ascorbic acid (Szent-Giorgyi A (1963) Lost in the twentieth century. Annu Rev Biochem 36:1–15). “Ascorbic” literally means “against scurvy”, and scurvy is known to be mainly due to the inactivation of some important dioxygenases involved in the synthesis of a few key molecules, including different collagen forms (De Tullio MC (2004) How does ascorbic acid prevent scurvy? A survey of the nonantioxidant functions of vitamin C. In: Asard H, May J, Smirnoff N (eds) Vitamin C, its functions and biochemistry in animals and plants. Bios Scientific Publishers, Oxford, UK, pp. 159–172). All this has very little to do with the celebrated role of ascorbic acid (ASC) as an antioxidant. So, if the fate of ASC had to be found in its name, its role in the prevention of scurvy (i.e. beyond the antioxidant function) should be considered its main feature. But, in spite of more than 80 years of extensive research (34,424 hits in a PubMed query on January 6 2007), an unprecedented popularity among the general public, an estimated market of several billion dollars (Hancock RD, Viola R (2005) Improving the nutritional value of crops through enhancement of l-ascorbic acid (vitamin C) content: Rationale and biotechnological opportunities. J Agr Food Chem 53:5248–5257), we should honestly conclude that the fate of vitamin C is still in the first name it received, many years ago: we still ignore much of its actual relevance in cell metabolism, although we are progressively getting aware of the many facets of this fascinating molecule, and its direct involvement in the regulation of apparently unrelated pathways (Arrigoni O, De Tullio MC (2002) Ascorbic acid, much more than just an antioxidant. Biochim Biophys Acta 1569:1–9; De Tullio MC, Arrigoni O (2004) Hopes, disillusions and more hopes from vitamin C. Cell Mol Life Sci 61:209–219; Duarte TL, Lunec J (2005) When is an antioxidant not an antioxidant? A review of novel actions and reactions of vitamin C. Free Rad Res 39:671–686). Recent data on ASC involvement in cell signalling and gene expression open new perspectives, that will be presented and discussed in this chapter.